Project Summary/Abstract: The 3D packaging of chromatin within the nucleus is thought to play an important role in regulating gene expression. It is often assumed that promoters, enhancers, and other critical regulatory elements exert influence by forming loops in DNA that bring key regulatory elements into close spatial proximity. However, recent studies have shown that DNA loops have a relatively minor impact on gene expression, challenging the generality of this paradigm. In fact, most experiments that investigate the relationship between gene expression and genome 3D structure rely on correlated measurements and usually lack direct evidence that spatial DNA interactions have an impact on gene expression. In contrast, we recently discovered that transcription elongation can directly impact genome 3D structure by displacing the ring-like molecule cohesin from chromatin, leading to the loss of DNA loops. These findings and other reports suggest that we lack an adequate understanding of how chromatin looping is involved in the execution of gene expression programs. A more detailed view of how genome 3D structure and transcription regulate one another is essential to interpret how regulatory programs are encoded in the genome, and has important consequences for studying genome function in nearly all areas of biomedical research. This proposal leverages recently developed reagents and sensitive profiling techniques to investigate how genome 3D structure impacts transcription and, reciprocally, how transcription impacts genome 3D structure in response to acute cellular stimulation. Using nascent initiation profiling (Start-seq), we will systematically characterize how loss of chromatin looping impacts inducible transcription at both promoter and enhancer elements and examine how perturbation of 3D structure impacts the functional communication between distal regulatory elements and their target genes. This proposal will also explore how transcriptional activation itself may regulate chromatin loop formation or dissociation as a direct consequence of changes in transcription. These studies will help reveal how chromatin 3D structure and transcription influence each other and will help improve the interpretation of regulatory programs encoded in the genome.